Clinical proteomics : a focus on transformed human T and B lymphocytes

Résumé large public La protéomique clinique est une discipline qui vise l'étude des protéines dans un but diagnostique ou thérapeutique. Nous avons utilisé cette approche pour étudier les lymphocytes T «tueurs » ou cytotoxiques qui font partie des globules blancs du système sanguin et agissent dans la lutte contre les infections et les tumeurs. Ces cellules sont impliquées dans l'immunothérapie cellulaire qui se fonde sur la capacité naturelle des ces lymphocytes à repérer les cellules tumorales et à les détruire. L'introduction du gène de la télomérase dans les lymphocytes T résulte en une prolongation de leur longévité, ce qui en ferait des candidats intéressants pour l'immunothérapie cellulaire. Il subsiste cependant des doutes quant aux conséquences de l'utilisation de ces lymphocytes «immortalisés ». Pour répondre à cette question, nous avons comparé le profile protéique de lymphocytes T cytotoxiques «jeunes » et vieux » avec celui des lymphocytes «immortalisés ». Nous avons trouvé que ces derniers présentent une double face et partagent à la fois les caractéristiques de la jeunesse et de la vieillesse. Dans une seconde étude de protéomique clinique, nous nous sommes penchés sur les lymphocytes B «immortalisés » cette fois-ci non pas avec la télomérase, mais avec le virus d'Epstein-Barr. Ces derniers sont utilisés comme modèle dans l'étude de la leucodystrophie, une maladie génétique rare qui affecte le cerveau. Notre but est d'identifier des marqueurs biologiques potentiels qui pourraient aider le diagnostic et le traitement de cette maladie neurodégénérative. Nous avons pour ce faire comparé les profiles protéiques des lymphocytes B «immortalisés » provenant d'individus sains et malades. Malheureusement, notre analyse n'a pas révélé de différences notoires entre ces deux classes de lymphocytes. Ceci nous permet toutefois de conclure que la maladie n'affecte pas la synthèse des protéines de manière prépondérante dans ces cellules sanguines. En résumé, le travail présenté dans cette thèse montre à la fois le potentiel et les limites de l'analyse des protéines lymphocytaires, dans différentes situations biologiques. Résumé La protéomique clinique ouvre la porte vers de multiples horizons relatifs au traitement de diverses maladies. Ce domaine particulier alliant la protéomique à la médecine, implique l'intervention de la biologie moléculaire et cellulaire. Dans notre étude, nous nous sommes d'abord intéressés aux lymphocytes T CD8+ cytotoxiques dans le contexte de l'immunothérapie adoptive. Le fondement de cette thérapie repose sur la capacité naturelle de ces lymphocytes à reconnaître les cellules tumorales et à les détruire chez les patients atteints de cancer. L'introduction du gène de la transcriptase réverse de la télomérase (hTERT) dans les lymphocytes T humains permet de rallonger leur durée de vie, sans toutefois induire d'altérations liées à la transformation. Cependant, des incertitudes subsistent quant à la ressemblance physiologique et biochimique entre ces cellules surexprirnant la télomérase et les cellules normales. Afin de répondre à cette question, nous avons comparé l'expression des protéines de lymphocytes humains T CD8+ «jeunes » et «vieux »avec celle de lymphocytes transduits avec hTERT. Nous avons trouvé que les lymphocytes T surexprimant la télomérase ont un profile protéique intermédiaire, avec certaines expressions protéiques similaires aux jeunes cellules T et d'autres se rapprochant des cellules vieilles. Dans la seconde partie de notre étude, nous nous sommes intéressés aux lymphocytes B transformés avec le virus d'Epstein-Barr provenant de patients atteints d'une maladie génétique rare du cerveau, la leucodystrophie. Dans cette maladie, des mutations dans le facteur de transcription eIF2B, impliqué dans la synthèse protéique, ont été trouvées. Afin d'analyser les conséquences de ces mutations et de trouver des biomarqueurs spécifiques à cette maladie, nous avons effectué une analyse protéomique des lymphoblastes provenant de malades et d'individus sains. Nous avons trouvé que les mutations dans le complexe ubiquitaire eIF2B n'affectent pas de manière significative l'expression des protéines des lymphoblastes mutés. En conclusion, notre travail illustre le potentiel et les limitations des technologies protéomiques utilisées pour disséquer l'implication des protéines dans différentes situations biologiques. Summary Clinical proteomics opens the door to multiple applications related to the treatment of diseases. This particular field is at the crossroad of proteomics and medicine and involves tools from cellular and molecular biology. We focused first our investigations on cytotoxic T cells in the context of adoptive immunotherapy, which is an interesting and evolving field. The basis of this therapy relies on the natural capacity of cytotoxic CD8+ T lymphocytes in recognizing tumor cells and destroying them in cancer patients. As their number is reduced, the idea would be to use transformed T lymphocytes with extended life span. Overexpression of telomerase into human T lymphocytes results in the extension of their replicative life span, but it still remains unclear whether these cells are physiologically indistinguishable from normal ones. To address this question, we compared the proteome of young and aged CD8+ T lymphocytes with that of T cells transduced with hTERT and found that the latter cells displayed an intermediate protein pattern, sharing similar protein expression with young, but also with aged T cells. We were then interested in studying Epstein-Barr virus transformed B lymphocytes in the context of a rare human brain genetic disorder called leukodystrophy. In this disease, mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported. In order to analyze the functional consequences of the mutations and to find out specific biomarkers of eIF2B-related disorders, proteomic and peptidomic studies were carried out on lymphoblasts from eIF2Bmutated patients versus healthy patients. Following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not appear to significantly affect the proteome of the mutated lymphoblasts extracts. To conclude, our work emphasizes the potentials and the limitations of the proteomic technologies used to analyze the role of lymphocyte proteins in different biological situations.

T and B lymphocytes exert distinct effects on the homeostasis of NK cells.

There is growing evidence that lymphocytes impact the development and/or function of other lymphocyte populations. Based on such observations we have tested whether the NK cell compartment was phenotypically and functionally altered in the absence of B and/or T cells. Here we show that T cell deficiency significantly accelerates BM NK cell production and the subsequent seeding of splenic and liver NK cell compartments. In contrast, B cell deficiency reduces splenic NK cell survival. In the absence of T and B cells, the size of the NK cell compartments is determined by the combination of these positive and negative effects. Even though NK cell homeostasis is significantly altered, NK cells from T and/or B cell-deficient mice show a normal capacity to kill a susceptible target cell line and to produce IFN. Nevertheless, we noted that the usage of MHC class I-specific Ly49 family receptors was significantly altered in the absence of T and/or B cells. In general, B cell deficiency expanded Ly49 receptor usage, while T cell deficiency exerted both positive and negative effects. These findings show that B and T cells significantly and differentially influence the homeostasis and the phenotype of NK cells.

Notch signaling in T- and B-cell development.

The Notch family of evolutionarily conserved proteins regulates a broad spectrum of cell-fate decisions and differentiation processes during fetal and post-natal development. The best characterized role of Notch signaling during mammalian hematopoiesis and lymphopoiesis is the essential function of the Notch1 receptor in T-cell lineage commitment. More recent studies have addressed the roles of other Notch receptors and ligands, as well as their downstream targets, revealing additional novel functions of Notch signaling in intra-thymic T-cell development, B-cell development and peripheral T-cell function.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Expression and presentation of endogenous mouse mammary tumor virus superantigens by thymic and splenic dendritic cells and B cells.

Tolerance against superantigens (SAgs) encoded by endogenous mouse mammary tumor virus (Mtv) loci involves the intrathymic deletion of SAg-reactive T cells expressing a particular TCR V beta-chain, presumably upon presentation of the SAg by specialized APC. However, although the role of dendritic cells (DC) in the induction of tolerance against conventional Ags has been demonstrated, little is known about the role played by DC in tolerance induction against Mtv SAgs. Moreover, there is conflicting evidence concerning the capacity of DC to express and present Mtv SAgs. In this report we have analyzed the expression of Mtv SAgs in highly purified thymic and splenic DC and B cells by reverse transcriptase-PCR, using primers amplifying Mtv SAg-specific spliced mRNAs. DC express Mtv SAgs at levels comparable to B cells, but display a differential expression pattern of the various Mtv loci compared with B cells. Furthermore, our results show that DC are able to induce the deletion of SAg-reactive thymocytes in an in vitro assay, indicating that Mtv SAgs are functionally expressed on the DC surface. Collectively, our data are consistent with the hypothesis that DC play a role in the induction of intrathymic tolerance to Mtv SAgs.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells.

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus.

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.

Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma.

Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas.

Simultaneous B(0)- and B(1)+-map acquisition for fast localized shim, frequency, and RF power determination in the heart at 3 T.

High-field (>or=3 T) cardiac MRI is challenged by inhomogeneities of both the static magnetic field (B(0)) and the transmit radiofrequency field (B(1)+). The inhomogeneous B fields not only demand improved shimming methods but also impede the correct determination of the zero-order terms, i.e., the local resonance frequency f(0) and the radiofrequency power to generate the intended local B(1)+ field. In this work, dual echo time B(0)-map and dual flip angle B(1)+-map acquisition methods are combined to acquire multislice B(0)- and B(1)+-maps simultaneously covering the entire heart in a single breath hold of 18 heartbeats. A previously proposed excitation pulse shape dependent slice profile correction is tested and applied to reduce systematic errors of the multislice B(1)+-map. Localized higher-order shim correction values including the zero-order terms for frequency f(0) and radiofrequency power can be determined based on the acquired B(0)- and B(1)+-maps. This method has been tested in 7 healthy adult human subjects at 3 T and improved the B(0) field homogeneity (standard deviation) from 60 Hz to 35 Hz and the average B(1)+ field from 77% to 100% of the desired B(1)+ field when compared to more commonly used preparation methods.

Response-to-comments about: "Is it really the method for carbon dioxide determination in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry valid?" from T. Saffaj and B. Ihssane

Saffaj et al. recently criticized our method of monitoring carbon dioxide in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry. According to the authors, their demonstration, based on the latest SFSTP guidelines (established after 2007 [1,2]) fitted for the validation of drug monitoring bioanalytical methods, has put in evidence potential errors. However, our validation approach was built using SFSTP guidelines established before 2007 [3-6]. We justify the use of these guidelines because of the post-mortem context of the study (and not clinical) and the gaseous state of the sample (and not solid or liquid). Using these guidelines, our validation remains correct.

A novel and divergent role of granzyme a and B in resistance to helminth infection.

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA×B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Spatially and genetically distinct control of seed germination by phytochromes A and B.

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.